RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth - PubMed (original) (raw)

RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth

Sateesh Kunigal et al. Int J Cancer. 2007.

Retraction in

Abstract

The serine protease urokinase-type plasminogen activator (uPA) plays a significant role in tumor cell invasion and metastasis when bound to its specific receptor, uPAR (also known as CD87). In addition to the uPA-uPAR system, matrix metalloproteinases (MMPs) are involved in tumor cell invasion and metastasis. In this study, we achieved specific inhibition of uPAR and MMP-9 using RNAi technology. We introduced small interfering RNA to downregulate the expression of uPAR and MMP-9 (pUM) in breast cancer cell lines (MDA MB 231 and ZR 75 1). In vitro angiogenesis studies indicated a decrease in the angiogenic potential of the treated cells; in particular, a remarkable decrease was observed in the cells treated with bicistronic construct (pUM) in comparision to the controls. Additionally, bicistronic construct inhibited the formation of capillary-like structures in in vivo models of angiogenesis. Similarly, the invasive potential and migration decreased dramatically when treated with the bicistronic construct as shown by matrigel invasion and migration assays. These results suggest a synergistic effect from the simultaneous downregulation of uPAR and MMP-9. We also assessed the levels of phosphorylated forms of MAPK, ERK and AKT signaling pathway molecules and found reduction in the levels of these molecules in cells treated with the bicistronic construct as compared to the control cells. Furthermore, targeting both uPAR and MMP-9 totally regressed orthotopic breast tumors in nude mice. In conclusion, our results provide evidence that the simultaneous downregulation of uPAR and MMP-9 using RNAi technology may provide an effective tool for breast cancer therapy.

(c) 2007 Wiley-Liss, Inc.

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Figures

Figure 1

Figure 1. Inhibition of MMP-9 Activity and uPAR Protein Levels by RNA Interference

MDA MB 231 and ZR 751 cells were transfected with CMV promoter-based vectors: control, an empty vector, and a vector encoding either single or bicistronic siRNA for uPAR and MMP-9 (puPAR, pMMP-9, and pUM). (A) MMP-9 activity was measured in conditioned media (40 μg) from SV-, puPAR-, pMMP-9-, and pUM-transfected cells using gelatin zymography. Densitometric analysis was done for MMP-9. (B) Western blot analysis of uPAR protein expression in cell lysates from MDA MB 231 and ZR 751 cells transfected with SV, puPAR, pMMP-9, and pUM. Western blot analysis was performed using an antibody specific for uPAR. GAPDH was simultaneously immunodetected to verify equal loading of cell lysates. Densitometric analysis was done for uPAR expression. The result provided is a representative experiment repeated 3-4 times with concordant results.

Figure 2

Figure 2. RNA Interference Inhibited uPAR and MMP-9 Immunofluorescence and Tumor-induced Angiogenesis

(A) MDA MB 231 and ZR 751 cells were transfected with SV, puPAR, pMMP-9, and pUM. Control (untransfected) cells were also used. 72 h after transfection, the cells were fixed and stained for uPAR and MMP-9 expression. The cells were mounted using mounting media with 4’,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus. (B) Conditioned media from MDA MB 231 and ZR 751 cells transfected with SV, puPAR, pMMP-9, and pUM were collected. Human microvascular endothelial cells (HMEC) (8×103 cells) were cultured in the conditioned medium collected in 8 well-chambered slides for 12-18 h. After the incubation period, the medium was removed and the cells were stained with HEMA-3 stain and examined under microscope. (C) Quantification of angiogenesis in co-cultures transfected with control, SV, puPAR, pMMP-9, and pUM. Values are mean ± S.D. from four different experiments. (D) Inhibition of tumor angiogenesis by pUM as assessed by mouse dorsal window assay. PV, pre-existing vasculature; TN, tumor-induced vasculature.

Figure 3

Figure 3. siRNA for uPAR and MMP-9 Inhibits Invasion of MDA MB 231 and ZR 751 cells

MDA MB 231 and ZR 751 cells (1×106) transfected with SV, puPAR, pMMP-9, and pUM were allowed to migrate through matrigel-coated transwell inserts (8-μm pores) for 24 h. (A) The cells that invaded through the matrigel-coated inserts were stained, counted, and photographed under a light microscope at 20X magnification. **(B)**The percentage of invasion was quantified as described in Materials & Methods. Values are mean ± S.D. from five different experiments (p<0.001). (C) We selected MDA MB 231 and ZR 751 cells spheroids that were intact and of approximately the same diameter. Then, the spheroids were transfected with SV, puPAR, pMMP-9 and pUM and incubated for 72 h to allow for migration. Finally, cell migration was observed using a confocal laser-scanning microscope after application of HEMA stain. The result provided is a representative experiment repeated 3-4 times with concordant results.

Figure 4

Figure 4. Therapeutic Effect of siRNA for uPAR and MMP-9

MDA MB 231 cells (5-6×106 cells in 100 μL of phosphate-buffered saline or serum-free medium) were injected into the breast pad of nude mice and kept under observation for tumor growth. Tumors were allowed to grow until they reached 5 to 6 mm. At this point, tumors were randomized into several groups receiving treatment with SV, puPAR, pMMP-9, or pUM (150 μg of each vector was injected intratumorally). (A) Animals injected with various constructs. (B) Tumors from these animals were dissected 4 weeks after injection with these constructs. (C) Semiquantification of tumor volume in control, SV, puPAR, pMMP-9, and pUM-treated groups at 4 to 6 weeks after treatment. Data shown are mean ± S.D. values from 5 to 6 animals from each group. (D) Tumor sections stained with hematoxylin and eosin.

Figure 5

Figure 5. siRNA against uPAR and MMP-9 Inhibits the Levels of Phosphorylated ERK, MAPK, and AKT

Western blot analyses of total and phosphorylated forms of p38 MAPK, ERK, and AKT. MDA MB 231 and ZR 751 cells were transfected with SV, puPAR, pMMP-9 and pUM, lysed 72 h later, and subjected to SDS-PAGE and immunoblotting with antibodies for total and phosphorylated forms of MAPK, ERK, and AKT. GADPH antibodies were used to verify that similar amounts of protein were loaded in each lane. Densitometric analysis of phospho- molecules was done. The result provided is of a representative experiment repeated 3-4 times with concordant results.

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